High Mobility Group Box 1 (HMGB1) is a non-histone chromatin-binding protein that plays a major role in human health and diseases. The passive or active release of HMGB1 from necrotic, immune or stressed cells induces the translocation of nuclear factor-κB (NF-κB); this results in the secretion of tumor necrosis factor α (TNF-α) and other pro-inflammatory cytokines. Previous studies have indicated that TLR4 is required for the HMGB1-dependent activation and release of TNF from macrophages. Moreover, the activity and interaction between TLR4 and its ligands are dependent on the molecular collaboration with Myeloid Differentiation factor 2 (MD-2). Recent studies have indicated that HMGB1 specifically binds to TLR4/MD-2 in the presence of cysteine 106 and MD-2 specifically binds to the disulfide isoform of HMGB1. Despite the existence of 44% similarity between the pro-inflammatory (B-box) and anti-inflammatory (A-box) domains, these proteins carry out different functions during inflammatory responses. The B-box recapitulates the cytokine activity of full-length HMGB1 and evokes the release of TNF from macrophages while the A-box (which contains the epitope for the anti-HMGB1 monoclonal antibody, 2G7) inhibits HMGB1-induced pro-inflammatory cytokines release and acts as an antagonist of HMGB1. However, the mechanism for the inhibition of HMGB1 by the A-box is unknown. Also, the molecular mechanism through which 2G7 exerts anti-inflammatory activity has remained a mystery. Thus, this study aimed to investigate the interactions of HMGB1 isoforms (redox state) and fragments (A- and B-box) with the TLR4/MD-2 complex.
Recently, Drs Mingzhu He, Tom Coleman, Kevin Tracey and Professor Yousef Al-Abed at The Feinstein Institute for Medical Research in New York in collaboration with Dr. Marco Bianchi at San Raffaele University and San Raffaele Scientific Institute demonstrated that HMGB1 interacts with TLR4/MD-2 in a two-stage process and HMGB1 A-box domain alone antagonizes HMGB1 due to its higher binding affinity to TLR4. All experiments were conducted by Surface Plasmon Resonance (SPR). Their study is now reported in Molecular Medicine.
The research team observed that the disulfide HMGB1 isoform binds to TLR4/MD-2 with a relatively high affinity while the non-oxidizable 3S mutant and the DTT-reduced HMGB1 binds to TLR4/MD-2 complex with approximately 10-fold lower affinity. However, reduced HMGB1 binds to TLR4 with a comparable equilibrium dissociation constant with disulfide HMGB1, indicating that a disulfide bond between C23 and C45 has a negligible influence on the binding to TLR4.
The authors noticed that the GST-A-box had a high affinity to TLR4 compared to TLR4/MD-2. It interacted with both complexes at different dissociation rate constants. However, a weak binding activity was noticed on the MD-2 immobilized chip. They also observed that the association rate and affinity of MD-2 to GST-A-box were weak and low compared to TLR4 or TLR4/MD-2. They demonstrated that the major binding site(s) of A-box domain to TLR4/MD-2 are located on TLR4.
Contrastingly, the authors found that GST-B-box exhibited a quick association to TLR4/MD-2 complex, which dissociated quickly by washing. There was no binding activity between GST-B-box and immobilized TLR4. However, there was a concentration-dependent binding of MD-2 to immobilized GST-B-box. These data indicate that B-box alone binds to MD-2, but not to TLR4. Furthermore, a sequential dual-injection method reveals that HMGB1 likely activates TLR4 signaling through inducing TLR4/MD-2 complex formation; and demonstrates that A-box directly competes with HMGB1 for binding to TLR4.
This novel study not only showed that HMGB1 and its fragments (A-box and B-box) individually interact with the TLR4/MD-2 receptor with different binding and kinetic parameters but also explained the molecular mechanism through which the A-box domain and 2G7 inhibit the activity of HMGB1. The intricate findings by Mingzhu He and colleagues will advance further studies on the interactions between HMGB1/TLR4/MD-2 complexes and the development of therapeutics to inhibit HMGB1-mediated inflammation.
He, M., Bianchi, M.E., Coleman, T.R., Tracey, K.J., and Al-Abed, Y. Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance, Molecular Medicine (2018) 24:21, 1-9.Go To Molecular Medicine (2018)